Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.
Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.
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加帽反基因组 RNA 转录本有助于拯救植物弹状病毒
DOI：
10.1186/s12985-017-0776-7
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发表时间：
2017-06-13
期刊：
影响因子：
4.8
通讯作者：
Li Z
中科院分区：
文献类型：
--
作者：
Qian S;Chen X;Sun K;Zhang Y;Li Z
从克隆的 cDNA 中回收重组负链 RNA 病毒是一个低效的过程，因为需要将多种病毒成分递送到细胞中以重建感染实体。此前的研究表明，真实的病毒RNA末端对于有效拯救病毒至关重要。然而，对于通过不同策略加工的病毒RNA在支持植物负链RNA病毒的恢复方面的活性知之甚少。在这项研究中，我们使用了锤头核酶的多个版本和截短的花椰菜花叶病毒 35S 启动子来生成桑葚黄网弹状病毒 (SYNV) 反基因组 RNA (agRNA) 衍生物的精确 5' 末端。这些 agRNA 与本塞姆氏烟草叶子中的 SYNV 核心蛋白共表达，以评估它们支持 SYNV 微型复制子 (MR) 荧光报告基因表达和拯救全长病毒的效率。锤头核酶裂解活性的优化提高了 SYNV MR 报告基因的表达。尽管由最活跃的锤头变体处理的 MR agRNA 在支持 MR 活性方面与加帽、精确转录的 agRNA 相当，但只有使用加帽 agRNA 才能实现重组 SYNV 的有效恢复。进一步的研究表明，加帽的SYNV agRNA允许核衣壳(N)蛋白的瞬时表达，而不能顺式表达N蛋白的agRNA衍生物表现出显着降低的救援效率。我们的研究揭示了精确转录的加帽 SYNV agRNA 的活性优于未加帽的锤头核酶加工的 agRNA，并表明在从质粒中回收 SYNV 的过程中，加帽 agRNA 表达的 N 蛋白具有顺式作用功能。本文的在线版本 (doi:10.1186/s12985-017-0776-7) 包含补充材料，可供授权用户使用。
Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5′ termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids. The online version of this article (doi:10.1186/s12985-017-0776-7) contains supplementary material, which is available to authorized users.